Winetech Technical Yearbook 2022

FIGURE 1. CRISPR/Cas system and repair mechanism for gene editing by targeting specific DNA sequences.

HOW DOES GENE EDITING WORK? Doudna and Charpentier demonstrated that the Cas protein can easily be re programmed to find and bind to almost any desired target sequence, simply by attaching a piece of RNA (guide RNA) to steer it in its search. When the Cas protein and a guide RNA are delivered into a cell, they form an editing complex that moves along the strands of DNA until it finds and binds to a 20-DNA-letter long sequence that matches part of the guide RNA sequence (figure 1). This is an impressive feat, considering that the DNA packed into each human cell has six billion letters and is two metres long. Once the target sequence has been recognised, the Cas protein cuts the DNA at the target site. The cut gets repaired by one of two natural repair mechanisms, the most common of which is error-prone, resulting in a knock out mutation that usually disable the target gene. Another repair mechanism relies on homology repair and can be used to

introduce foreign DNA, creating a knock in mutation. So, for instance, scientists could program the Cas protein to snip out a gene that causes Huntington’s disease in humans and insert a “good” gene to replace it. Numerous types of CRISPR/Cas systems have been discovered – the one we described so far is the most widely used and well known: CRISPR/Cas9. Several more Cas proteins have been characterised that have slightly different mechanisms and target requirements. For example, Cas13, can cut RNA instead of DNA. ADVANTAGES AND DISADVANTAGES OF THE TECHNOLOGY Various techniques to knock out genes have been around for years. What makes CRISPR so revolutionary is that it’s so precise: the Cas protein mostly goes wherever it is directed. And, as mentioned before, it’s incredibly versatile, cheap and easy. Also very promising, this technique

has worked on every organism it’s been tried on: animals, plants and bacteria. It provides the possibility to cure genetic diseases and to produce plants that can resist pathogens and adverse climate conditions. CRISPR can be used for DNA free gene editing without the use of foreign DNA. A DNA-free gene editing system can be a good choice to avoid the possibility of undesirable genetic alterations due to the foreign DNA integrating randomly. In spite of being such a recent discovery, CRISPR technology has already achieved incredible results. However, each CRISPR system needs to be optimised to be accurate and reliable, especially in eliminating the so called ‘off-targets’ edits. Indeed, it has been observed that sometimes a sequence not exactly the same as the one intended, can be recognised and cut by the CRISPR/Cas. Like all powerful technologies, it is important that safety and ethical aspects are considered. Scientists must consider that there are potential hazards of using this technology in an irresponsible way.

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WINETECH TECHNICAL YEARBOOK 2022